G-protein Regulation of the Phosphatidyl Inositol (3,4,5) Trisphosphate Signal

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DESCRIPTION (provided by applicant): G protein coupled receptors provide both positive and negative regulation of hematopoietic cells. In cells such as neutrophils, macrophages, mast cells and platelets, the p110gamma isoform of phosphatidylinositol (4,5) 3-kinase (Ptdlns 3-kinase) plays a major role in cell activation, shape changes and migration via generation of phosphatidylinositol (3,4,5) trisphosphate (PIP3). This lipid is an important signal that activates the phosphatidylinositol dependent protein kinase, PDK-1, leading to phosphorylation of protein kinase B and a host of cell responses. The levels of PIP3 in the membrane of these cells are also tightly controlled by a SH2 domain containing inositol 5-phosphatase, SHIP, that is regulated by multiple mechanisms. Activation of G protein coupled receptors linked to Gi stimulates the p110gamma isoform of Ptdlns 3-kinase 40-60 fold by releasing specific betagamma dimers. Activation of G protein coupled receptors linked to Gs raise cyclic AMP and can markedly inhibit the response of hematopoietic cells to stimulatory ligands. Our research has provided clear evidence of the specific isoforms of the betagamma dimer which activate the p110gamma isoform of Ptdlns 3 kinase. Recent experiments uncover the exciting result that both the p110gamma isoform of Ptdlns 3-kinase and SHIP can be phosphorylated by the cyclic AMP dependent protein kinase. The ability of the betagamma dimer to activate p101/p110gamma is inhibited by phosphorylation. Phosphorylation of SHIP activates the enzyme. These results provide a possible molecular explanation for the ability of cyclic AMP to inhibit the response of hematopoietic cells. The goal for this project is to determine the importance of these phosphorylation events in cell function. This goal will be approached via 2 Specific Aims. Aim-1a: To determine the effects of phosphorylation on the activity of Ptdlns 3-Kinase in vitro. Aim-1b: To understand how phosphorylation of Ptdlns 3-Kinase regulates the function of the enzyme in three cell lines. Aim-2a: To explore the effects of phosphorylation on the activity of SHIP in vitro. Aim-2b: To understand how phosphorylation of SHIP regulates its function in the intact cell. Completion of these Aims will provide considerable understanding of the molecular events regulating the levels of PIP3 in all cells and should reveal mechanisms by which G protein coupled receptors stimulate and inhibit the function of hematopoietic cells.